Method of inducing cell proliferation using fibroin

ABSTRACT

A bioactive material is made using fibroin solutions and suspensions designed to support the constructions, repair, regeneration or augmentation of bone and other tissues of the body. The fibroin solutions, suspensions, and composites can be injected to fill cavities or to replace missing tissue. After injection, the materials can produce a scaffold capable of promoting tissue regeneration while degrading. The ability to inject the fibroin solutions, suspensions and composites can reduce the need for many types of surgical procedures that are used to replace or repair bone and other tissues. The fibroin solutions and suspensions are particularly useful for methods of tissue construction and/or repair.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/281,096 filed Oct. 25, 2002, which claims priority from ProvisionalApplication Ser. No. 60/343,006, filed Oct. 25, 2001, both incorporatedherein by reference in their entirety.

BACKGROUND

This disclosure relates to the field of bone and tissue repair, and inparticular to materials for bone and tissue repair, methods of makingthe materials, and methods for use of the materials.

Replacement and repair of bone and other tissues following injury oftenrequires the use of surgical procedures. More than 300,000 hipprostheses are implanted each year in the United States and Europe.Additionally, 10% of the population suffers from periodontal disease,and 30% will require a tooth implant during his/her lifetime. Othersurgical procedures include cartilage repair and plastic surgery of softtissues. It is thus desirable to create scaffold materials for tissuerepair or reconstruction, particularly injectable materials that caneliminate the need for many invasive procedures. Such materials shouldbe biocompatible, i.e., not cytotoxic or causing adverse reaction in thebody and preferably bioactive, i.e., providing the developmental signalsneeded for mobilization of the cell activity required for tissuebuilding. They are furthermore preferably resorbable, and capable ofwithstanding the stresses imposed by daily activity during repair.

Several different approaches to scaffolds for tissue repair have beensuggested. Currently available materials for hard tissue repair such asdemineralized bone, hydroxyapatite, tricalcium phosphates, and otherinorganic materials are not as effective as biologically derivedbioactive scaffolds. Hyaluronic acid has been used as a scaffoldmaterial as disclosed in U.S. Pat. No. 5,939,323. Another approach hasbeen to use collagen-based materials as disclosed in U.S. Pat. No.4,490,984 and U.S. Pat. No. 5,480,644. These materials, however, appearto be limited in their range of potential uses and applications becauseof poor mechanical properties, unpredictable degradation rates and, forcollagen, the risk of immunogenic reactions and dangers related topotential contamination.

Membranes, films and fabrics containing fibroin, a protein component ofsilkworm silk, have been suggested as substrate materials for the growthof animal tissues and organs. In particular, PCT Application number WO01/25403 describes the formation of fibroin membranes cast from watersolution. The membranes were cast in containers into which growth mediumand various cell types were added, and the fibroin membrane supportedthe growth of cells such as osteoblasts, epithelial cells, andhepatocytes. Fibroin membranes and films are also disclosed by Tsukadaet al., in Journal of Polymer Science: Part B: Polymer Physics 32:961-968, 1994; and by Motta et al., in “Third International Symposium onFrontiers in Biomedical Polymers Including Polymer Therapeutics FromLaboratory to Clinical Practice”, Abstract, Shigha Japan, May 1999. PCTApplication WO 02/29141 describes the formation of fibroin non-wovenfabrics made by treating fibroin cocoons with formic acid. The fibroinnon-woven fabrics can be used to culture cells such as keratinocytes andfibroblasts. A drawback of using such fibroin membranes, films, ornon-woven fabrics as a scaffolds for tissue repair in vivo is thatinvasive surgical procedures would be required in order to place thematerials at the site to be restored.

Accordingly, there remains a need for bioactive scaffold materials foruse both in vitro and in vivo, particularly materials that arebiocompatible, bioactive, and resorbable. There further remains a needfor bioactive scaffold materials that are readily applied to the site tobe restored, preferably without use of invasive surgical procedures.

SUMMARY OF THE INVENTION

The above discussed and other drawbacks and deficiencies of the priorart are overcome or alleviated by composition comprising a fibroinsolution or suspension. The fibroin solutions and suspensions arebiocompatible, bioactive, and resorbable. In a particularly advantageousfeature, the fibroin solutions or suspensions can be applied to adesired site by injection, which minimizes use of invasive surgicalprocedures.

Also disclosed is a method for the formation of a fibroin suspension,comprising heating a fibroin solution from about −20° C. to about 50° C.to form a fibroin suspension. Another method for the formation of afibroin suspension comprises treating a fibroin solution with an agenteffective to form a fibroin suspension. Effective agents comprise anacid, an alcohol, a proteolytic enzyme, a biocompatible polymer, orcombinations comprising at least one or more of the foregoing agents.

Further disclosed is a method of tissue construction, comprisingadministering to a site to be constructed a fibroin solution orsuspension in an amount effective to stimulate cell proliferation, andpreferably tissue growth. Administration is preferably by injection. Theabove discussed and other features and advantages will be appreciatedand understood by those skilled in the art from the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the exemplary drawings wherein like elements arenumbered alike in the several FIGURES:

FIG. 1 is a schematic diagram of a multi-component scaffold in theprocess of tissue repair.

FIG. 2 is an environmental scanning electron micrograph of a fibroinsuspension made by adding a water solution of citric acid to afibroin-water solution.

FIG. 3 is an environmental scanning electron micrograph of a fibroinsuspension made by adding glycerol to fibroin-water solution.

FIG. 4 is an optical micrograph of a fibroin composite suspensioncomprising hydroxyapatite/(poly)lactide core/shell particles embedded ina fibroin matrix after the composite was dried at room temperature.

FIG. 5 is an optical micrograph of a fibroin composite suspensioncomprising hydroxyapatite/(poly)lactide core/shell particles embedded ina fibroin matrix after the composite was dried at room temperature, athigher magnification.

FIG. 6 is an optical micrograph of a fibroin composite suspensioncomprising hydroxyapatite/(poly)lactide core/shell particles embedded ina fibroin matrix after the composite was freeze-dried.

FIG. 7 is an optical micrograph of a fibroin composite suspensioncomprising hydroxyapatite/(poly)lactide core/shell particles embedded ina fibroin matrix after the composite was freeze-dried, at highermagnification.

FIG. 8 compares the histologies after one month of implantation in acavity drilled in the femur of a rabbit and filled by an injectablefibroin suspension (A) to that of an empty cavity used as a control (B).

FIG. 9 compares the histologies after one month of implantation in acavity drilled in the femur of a second rabbit filled by an injectablefibroin suspension (A) to that of an empty cavity used as a control (B).

FIG. 10 shows the interface between the old bone and the new bone formedin the fibroin suspension filled cavity of the implant illustrated inFIG. 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Fibroin is a known polypeptide containing a combination of 18 differentamino acids, with glycine, alanine, serine, and tyrosine making upapproximately 90% of the polypeptide chain. It is generally acceptedthat fibroin consists of two major chains linked by a disulfide bond andhaving molecular weights of approximately 350,000 daltons (H-chain) and25,000 daltons (L-chain). The available studies of fibroin show that thestructure and morphology of manufactured products derived from fibroinare highly dependent on the processing conditions used to form them. Ithas been advantageously discovered herein that novel fibroin suspensionscan be produced from fibroin solutions using a variety of techniques,and that the fibroin suspensions so produced have utility in tissueconstruction, including the formation of tissue at normal sites in thebody (i.e., sites without injury) and sites in need of repair and/orreconstruction due, for example, to injury or aging. As used herein, a“fibroin solution” refers to composition having substantially one phase,that is, a composition comprising a solvent in which fibroin issubstantially dissolved. Fibroin is substantially dissolved when greaterthan 95%, preferably greater than 98%, and most preferably greater than99% of the fibroin, by weight, is in solution. Further as used herein, a“fibroin suspension” refers to a composition having two or more phases(i.e., a multi-phase material), with at least one phase comprising asolvent and at least one phase comprising fibroin. Without being held totheory, fibroin suspensions may exist in a variety of forms, forexample, in the form of a colloid, an emulsion, as micelles, a sol or agel.

For the purposes of describing the fibroin suspensions herein, referencewill be made to “fibroin occlusions”, “fibroin gels”, fibroin creams”,and “fibroin pastes”, which may be characterized by readily observablephysical characteristics such as appearance and relative viscosities.“Fibroin occlusion” refers to a fibroin suspension that is flowable on alevel surface. Occlusions are often cloudy, i.e., exhibit some opacity.By flowable on a level surface, it is meant that a one cubic centimetersample of the suspension will essentially immediately deform whendeposited on a level horizontal surface. Without being held to theory,fibroin occlusions may be in the form of colloids, particularly sols,comprising dispersions of solid particles having dimensions of 10⁻⁹ to10⁻⁶ meters in a continuous phase of the solvent.

A “fibroin gel” as used herein refers to a fibroin suspension that hasgel-like physical characteristics, for example, plasticity, elasticity,or some degree of rigidity. Gels can be opaque or translucent, dependingon the method used to form the gel. In contrast to an occlusion, a geldoes not readily flow when placed on a level surface. A gel, however,can flow or deform when heat or mechanical stress, such as pressure, areapplied, i.e., when the gel is reversible. Without being held to theory,a fibroin gel may contain a three-dimensional network of fibroindispersed in the solvent.

A “fibroin cream” as used herein refers to a fibroin suspension that ismore viscous than a fibroin occlusion. It is often white, and it issubstantially non-flowable on a level surface. By substantiallynon-flowable on a level surface, it is meant that a one cubic centimetersample of the fibroin cream will not appreciably deform within oneminute of being deposited on a level, horizontal surface. Like fibroingels, fibroin creams can deform when heat or mechanical stress areapplied to the material.

A “fibroin paste” as used herein refers to a fibroin suspension that ishighly viscous, and non-flowable when placed on a horizontal surface. Bynon-flowable on a level surface, it is meant that a one cubic centimetersample of the fibroin paste will not appreciably deform within one hourof being deposited on a level, horizontal surface. The paste isdeformable, however, under mechanical pressure.

Alternatively, the various types of fibroin suspensions may be describedbased on the injectability of the suspension through an aperture (i.e.,of a needle) of a particular size. A fibroin occlusion or gel is readilyinjectable by hand through a small aperture needle such as a 20-gaugesyringe needle having a diameter of about 584 micrometers. The fibroincream is more viscous, and is therefore readily injectable by handthrough a larger bore syringe needle, i.e., an 18 gauge needle having adiameter of about 838 micrometers. Pastes are not readily injectable byhand except through very large bore syringe needles, i.e., those havinga diameter of greater than about 2 millimeters. It is to be understoodthat the classification of a fibroin suspension as an occlusion, gel,cream, or paste is for convenience only in describing use of fibroinsuspensions.

The fibroin suspensions can be formed in vitro or in vivo. In vitroformation of fibroin suspensions can comprise treating a fibroinsolution with an agent effective to form the suspension, such as heat,proteolytic enzymes, acids, alcohols, or a biocompatible polymer.Alternatively, fibroin suspensions can be formed in vivo by injecting orotherwise administering to an animal a fibroin solution. Without beingheld to theory, it is believed that after administration to a site inthe body of an animal, a fibroin solution can rapidly dissipate excesswater, thereby producing a suspension in the form of, for example, agel. Fibroin suspensions can also undergo form changes in vivo. Forexample, a fibroin occlusion, after in vivo administration to a site inthe body of an animal, can undergo a transition to form a fibroin gel.

A convenient source of fibroin is cocoons from the Bombyx mori silkwormwhich contain both fibroin and sericin proteins. Silks are fibrousproteins produced from spiders and different insects, the best known ofwhich are silkworms (in particular the Bombyx-mori silkworm). Silkfibers have been used as sutures, but it has been found that braidedsilk sutures often produce a non-immunologic foreign-body reaction,causing granulomas even years after surgery (Kurosaki et al., Nippon IkaDaigaku Zasshi 66: 41-44, 1999). It has been confirmed, however, thatthe observed non-immunologic foreign-body reaction is caused by thepresence of sericin in the native silk, and that pure fibroin does notprovoke an immunological response.

Preferably, the fibroin is purified to remove toxins or other substancessuch as sericin that can cause adverse reactions in the body. Much ofthe sericin can be removed, for example, by degumming, i.e. washing insodium carbonate with or without sodium dodecyl sulfate at 98° C.

The purified fibroin can then be dissolved in a solvent such as anaqueous solution of lithium bromide containing approximately 10% byweight of fibroin per unit volume. The fibroin solution may optionallycontain other components such as buffers and other additives that do notsignificantly adversely affect the structure or stability of fibroin.The fibroin can be further purified preferably to greater than about90%, more preferably greater than about 95%, and most preferably greaterthan about 99% by weight. The Lithium Bromide is removed by dialysisagainst distilled water and other impurities may be removed byfiltration. Once purified, the fibroin can be freeze-dried to a powderand stored. Fibroin suspensions can then be formed from a pure fibroinsolution by treating the solution with heat, proteolytic enzymes, acids,alcohols, or a biocompatible polymer.

In one embodiment, fibroin suspensions can be produced by thermaltreatment of a fibroin solution. For instance, to produce a gel, thefibroin solution may be kept at about −20° C. for about 2 to about 24hours, then heated to a temperature of about 4° C. to about 50° C.Modification of the thermal treatment conditions results in suspensionsof varying attributes as described above, which can be selected by oneof ordinary skill in the art based on the particular application.

In another embodiment, a fibroin suspension can be produced by treatingthe fibroin solution with proteolytic enzymes that are specific forcleavage between particular amino acids. Such proteolytic enzymesinclude but are not limited to the protease from Streptomyces griseus,papain, chymotrypsin, and the like. Without being bound by theory, it ishypothesized that proteolytic enzyme treatment can produce a suspensionby reducing the average molecular weight of the fibroin polymer.Specific amino acid sequences can be selected to produce fragmentshaving desired molecular weights and amino acid sequences. The fragmentsizes can be tailored to meet specific demands of particularapplications, such as bioactivity and degradation rate of the fibroinmaterial. Selection of a particular average molecular weight will dependon a number of factors, including end use, desired viscosity, additionof other components, molecular weight distribution, type of carrier, andthe like. The average molecular weights of the fibroin after proteolytictreatment are about 200 to about 0.1 Kilodalton (Kd), preferably about50 to about 0.2 Kd, more preferably about 20 to about 0.5 Kd.

In another embodiment, the fibroin suspension can be formed by treatinga fibroin solution with an acid. Without being bound by theory, it isbelieved that by reducing the pH of the fibroin solution below itsisoelectric point, the miscibility of the fibroin in the solvent will bereduced, thus promoting suspension formation. The acid is an acidcapable of reducing the pH point of fibroin below its isoelectric pointof pH 3.8 (i.e., less than or equal to about pH 3.7). Preferred acidsare citric acid, ascorbic acid, lactic acid, combinations of theforegoing acids, and the like. The amount of acid added is that which issufficient to reduce the pH of the solution to the desired pH, and canbe readily determined by one of skill in the art.

The fibroin suspension can alternatively be formed by treating a fibroinsolution with an alcohol. Without being bound by theory, it is believedthat the alcohol can alter the fibroin protein conformation, thuspromoting formation of a suspension. The alcohol is preferably misciblewith the solvent, and can be, for example, glycerol, ethylene glycol,ethanol, isopropanol, and mixtures comprising one or more of theforegoing alcohols, and the like. A preferred alcohol is glycerol. Whenadded to the fibroin solution, the alcohol is added in an amount ofabout 10 volume % to about 50 volume % of the total volume.

In yet another embodiment, a more viscous fibroin solution can be formedby mixing a fibroin solution with one or more biocompatible polymerssuch as, for example, polyethyleneglycol, polyethyleneoxide,polyvinylpyrrolidone, and the like. The biocompatible polymers should bemiscible with the fibroin solution in an amount of about 2 to about 20%by weight.

Various additives can be used to improve the efficacy of the fibroinsolutions and suspensions, for example, physiologically active agentsthat have a physiological activity such as a diagnostic or therapeuticactivity. Accordingly, an active agent can include a detectable label(e.g., a radioactive label) that is useful for identifying the locationsof the released agent in vivo. Active agents also include therapeuticagents that are useful for treating a disease or condition.Physiologically active agents include, for example, antibiotics or othercompounds that inhibit infection; therapeutic agents for treatingosteoporosis, other factors that act on bone and skeleton, bonemorphogenetic proteins (BMPs), and other cytokines and the like thatstimulate tissue growth, bone regeneration or wound healing. Thephysiologically active agent can also be a cell, for example a celltaken from a site of the patient and cultured on the fibroin suspension.

Exemplary antibiotics include tetracycline, aminoglycosides,penicillins, cephalosporins, sulfonamide drugs, chloramphenicol sodiumsuccinate, erythromycin, vancomycin, lincomycin, clindamycin, nystatin,amphotericin B, amantidine, idoxuridine, p-amino salicyclic acid,isoniazid, rifampin, antinomycin D, mithramycin, daunomycin,chlorexidine, adriamycin, bleomycin, vinblastine, vincristine,procarbazine, imidazole carboxamide, and the like.

Exemplary therapeutic agents for treating osteoporosis and other factorsacting on bone and skeleton include calcium, alendronate, bone GLapeptide, parathyroid hormone and its active fragments, histoneH4-related bone formation and proliferation peptide and mutations,derivatives and analogs thereof.

Exemplary cytokines include transforming growth factors (TGFs),fibroblast growth factors (FGFs), platelet derived growth factors(PDGFs), epidermal growth factors (EGFs), connective tissue activatedpeptides (CTAPs), osteogenic factors, and biologically active analogs,fragments, and derivatives of such growth factors. Members of thetransforming growth factor (TGF) supergene family, which aremultifunctional regulatory proteins, are particularly preferred. Membersof the TGF supergene family include the beta transforming growth factors(for example TGF-beta 1, TGF-beta 2, TGF-beta 3); bone morphogeneticproteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9); heparin-binding growth factors (for example, fibroblastgrowth factor (FGF), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (forexample, Inhibin A, Inhibin B); growth differentiating factors (forexample, GDF-1); and Activins (for example, Activin A, Activin B,Activin AB). Growth factors can be isolated from native or naturalsources, such as from mammalian cells, or can be prepared synthetically,such as by recombinant DNA techniques or by various chemical processes.In addition, analogs, fragments, or derivatives of these factors can beused, provided that they exhibit at least some of the biologicalactivity of the native molecule. For example, analogs can be prepared byexpression of genes altered by site-specific mutagenesis or othergenetic engineering techniques.

The physiologically active agent can also be a differentiated ornon-differentiated cell. Mesenchymal stem cells, for example, can bedelivered to produce cells of the same type as the tissue into whichthey are delivered. Mesenchymal stem cells are not differentiated andtherefore can differentiate to form various types of new cells due tothe presence of an active agent or the effects (chemical, physical,etc.) of the local tissue environment. Examples of differentiatedmesenchymal stem cells include osteoblasts, chondrocytes, andfibroblasts. Osteoblasts can be delivered to the site of a bone defectto produce new bone; chondrocytes can be delivered to the site of acartilage defect to produce new cartilage; fibroblasts can be deliveredto produce collagen wherever new connective tissue is needed; etc. Thecells or genes may be either allogeneic or xenogeneic in origin. Forexample, the cells can be from a species other than the host speciesthat have been genetically modified.

The physiologically active agents can simply be added to the fibroinsolutions and suspensions or covalently bound to another component.Alternatively, the physiologically active agents can be added incontrolled release form. One controlled release formulation contains theactive agent dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedby Kent et al. in U.S. Pat. No. 4,675,189. Additional slow releaseformulations will be apparent to the skilled artisan. See, for example,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinsoned., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, ControlledRelease of Biologically Active Agents, John Wiley &Sons, New York, 1987.

In another embodiment, the fibroin solutions and suspensions cancomprise organic, inorganic and/or protein inclusions of engineered sizeand chemical composition that affect factors such as porosity andbiodegradation. In a preferred embodiment, the fibroin solution and/orsuspension can further comprise particles such as core/shell particlesthat create additional porosity in the gel, thereby forming a scaffoldafter injection.

Exemplary organic or inorganic particles that can aid in bone growth andformation, include, for example, hydroxyapatite, tricalcium phosphate,marine animal derived particles such as corals and chitosans, and thelike.

A preferred pore-forming particle is a core/shell particle in which aninorganic core is surrounded by a biodegradable organic shell. Suchparticles can serve as both a temporary support structure and aninternal substrate for shaping a scaffold. The controlled biodegradationof the shells can provide additional porosity to the fibroin scaffold.Core/shell particles will be described in detail below.

The preferred core is an inorganic core. The composition, size, andshape of the inorganic core can be varied to meet the demands ofdifferent applications. For example, the core can be hydroxyapatite,tricalcium phosphate, or marine animal derived particles, such as coralsand chitosans, to aid in bone ingrowth. The cores can have dimensionsfrom about 1 nanometer to about 500 micrometers in their largestdiameter, and be in the form of spheres, plates, fibers, and the like.

An optional reactive layer may be disposed on the core to enhanceadhesion of the core to the polymer shell. Suitable reactive layersdepend on the composition of the core and the shell, and are known inthe art. A particularly useful class of compounds includes reactivetitanates, zirconates, and silanes, for example,gamma-glycidoxypropyltriethoxysilane.

The shell can comprise at least one biodegradable polymer. Thecomposition and molecular weight of the polymer or polymers can bechosen to obtain an appropriate rate of biodegradation as describedbelow. Suitable biodegradable polymer compositions for the shellcomprise one or more of polylactic acid, polyglycolic acid,polycaprolactone, polytrimethylcarbonate, polyethylene glycoldiacrylates, polyanhidrides, polyorthoesters, polyphosphazines,polyacetals, polyesters, polyureas, polycarbonates, polyurethanes,polyalpha-hydroxy acids, polyamides, polyamino acids, and otherbiodegradable polymers or copolymers with an appropriate biodegradationrate and that are biocompatible.

The biodegradable polymer can be either a preformed polymer or may bepolymerized at the time of deposition on the core. The biodegradablepolymer layer can be tethered to the reactive layer or the core itselfby methods known in the art, for example by making a slurry of the coreparticles in a dilute organic solution of the reactive monomers or thepolymer. In one embodiment, the monomers or the polymers of the shellare chemically modified to include end groups such as peptides that areinteractive with cells within the body, for example, end groups that areactive in stimulating tissue repair or bone regeneration. Such endgroups can include, for example, peptides containingarginine-glycine-aspartic acid sequences, signal molecules, and many ofthe previously described physiologically active agents such as, forexample, bone morphogenic proteins.

The biodegradable shell can be selected to provide a balance between thetemporal mechanical stability of the scaffold and the development ofadditional internal porosity. The volume ratio of the core/shellcomponents can vary widely, from about 95/5 to 5/95, with a preferredcore/shell ratio of 20/80.

After adsorption of the polymers on the core, a stabilizing agent forthe suspended solids may optionally be added. The core/shell slurry maybe mixed with a dilute aqueous solution containing a small amount, forexample from about 0.5 wt % to about 3 wt %, of high molecular weightpolyethylene glycol or polyvinyl alcohol.

When the core/shell particles are formed as a slurry, the slurry can bemixed with a calcium source, and optionally a phosphate source, to forma coating on the exterior of the polymer shell of the formed particles.Exemplary precursors for providing the calcium and optional phosphatesource are a buffered water-alcohol solution of a calcium salt (e.g.,calcium acetate), a calcium salt and a phosphoric acid ester, or aformulated plasma fluid. This calcium ion-doped outer coating can serveas the interface with the bioactive fibroin matrix.

The resulting core/shell particles are in a relatively stable suspensionin the aqueous phase. After drying, for example, freeze-drying, theparticles can be lightly milled to form a core/shell powder. Onceformed, the core/shell powder can then be mixed with the fibroinsolution or suspension. The water content of the mixture can be adjustedto maintain fluidity. The relative quantities of particles and fibroinsolution or suspension can vary depending on the particular application.Typically, the solution or suspension can contain about 10 vol % toabout 50 vol % (percent by volume), preferably about 30 vol % to about50 vol % of the particulate phase.

Once formed, the fibroin solutions and/or suspensions can be used invivo as a scaffold for tissue construction, or administered to ananimal, for example, a mammal such as a rabbit, dog, cat, horse, human,and the like for tissue construction. As used herein, “tissueconstruction” is intended to include formation of hard or soft tissuethat is not at the site of an injury, for example in plastic surgery, orrepair or reconstruction at a site of injury.

If the fibroin suspension is in the form of an occlusion or a gel, thefibroin suspension preferably is administered to a tissue constructionsite by injection. A fibroin gel for injection is a reversible gel,i.e., a gel that can revert back to a less viscous state under, forexample, the application of heat or mechanical stress. Mechanical stresscan include the stress applied to the gel during the injection process.Alternatively, if the fibroin suspension is in the form of a cream or apaste, the fibroin suspension can be applied by placing in a tissueconstruction site (i.e., a site in need of tissue repair) by means suchas surgical placement or by spreading (i.e., topical administration).Without being held to theory, it is believed that regardless of the modeof administration, once applied, the fibroin suspensions can form amatrix or scaffold that can permit the infiltration and growth of thecells used in tissue construction.

The fibroin solutions and suspensions can be provided to a practitionerready for injection or other forms of administration. When mixed withparticles, the characteristics of fibroin solutions and suspensions canbe selected at the time of injection using a one- or two-syringetechnique. In the one-syringe technique, the relative quantities ofparticles and fibroin solution or suspension are provided by thepractitioner depending on the application, then mixed, and placed in atraditional single syringe with needle and injected. Alternatively, theparticles and fibroin solution or suspension can be provided in separatecartridges and mixed in a mixing chamber prior to placement. Use of adevice that allows variable mixing during placement provides thepractitioner with the ability to vary the composition of the scaffold tobetter match the variation of properties at a given site (e.g., bone orsoft tissue). The water contents of the two components are chosen toallow rapid mixing and injection.

In use, the fibroin solutions and suspensions are administered to ahuman or animal for a variety of purposes, for example to fill cavities,replace missing tissue, or for soft tissue or articular repair. Thefibroin solution or suspension can be administered by, for example,injection or topical application (i.e., applied as a cream to the skin).Without being held to theory, in the case of a solution, it is believedthat the solution can rapidly dissipate excess water into the body untilquasi-equilibrium is established between the injected material and thesurrounding bodily fluids, thereby producing a suspension that can forma scaffold in vivo. In the case of an occlusion, it is believed thatupon placement in the body, the occlusion can undergo a transition suchas a sol-gel transition, thereby producing a gel that can be in the formof a scaffold. In all cases, it is preferred that the materials, onceinjected, are porous.

Without being held to theory, it is believed that when the injectedfibroin solution or suspension comprises core/shell particles,degradation of the polymeric shells over time can generate additionalporosity within the suspension, i.e., in the form of a gel, forming ascaffold that enhances infiltration and growth of the cells necessaryfor tissue repair. Once injected, for example, the fibroin can comprisea three-dimensional web (or honeycomb) in the form of a gel thatencapsulates the dispersion of core/shell particles. FIG. 1 shows aschematic of a hypothetical three-dimensional web in the process oftissue repair. The fibroin 1 is infiltrated by cells and the core/shellparticles 2 undergo at least partial degradation. The dimensions andstate of aggregation of the core/shell particles control the additionalporosity of the scaffold. The newly formed tissue 3 can undergoorganization in the pores. The composition and morphology of the fibroinmatrix are the primary factors controlling bioactivity.

A porous fibroin scaffold can promote cell adhesion, proliferation andactivation, while slowly degrading during the healing process.Furthermore, degradation of the fibroin can produce peptides withmolecular weights in the range of about 20 Kilodaltons or less that canbe released into the surrounding biological fluid thus enhancing themetabolic activity of the surrounding cells. Without being held totheory, it is further hypothesized that thermal or proteolytic enzymetreatment can increase the biodegradation rate of fibroin by exposingspecific active sites for eventual interaction with growth factors,pharmacological molecules, and peptides specific for mediation ofcellular adhesion.

The above-described fibroin solutions and suspensions can be used, forexample, in a variety of repair procedures for bones and tissues.Exemplary procedures include, but are not limited to, orthopedic,maxillofacial, dental, and general surgical procedures such as tumorresection and plastic surgery. For example, fibroin solutions andsuspensions can be used in the repair of bone fractures via adhesivebonding, and rehabilitation of bones implicated in osteoporosis andosteoarthritis, by rehabilitating the affected bones. Such materials canalso be used as bone cements (for prostheses, for example), to fill oraugment tissues, to modify tissues size or shape, in periodontalpockets, as stabilizers for tooth and articular implants, and asfillings for gaps generated between hip, knee, and other prostheses andbone in order to achieve prosthesis immobilization and promote boneregeneration. Fibroin solutions and suspensions can also be used, forexample, to fill pockets formed around the teeth of periodontalpatients, thus permitting partial bone regrowth and possibly reducingbacterial infection; and to fill the gaps between bone and an implantsuch as, for example, a hip or knee implant. Filling such a gap canpotentially lead to prosthesis stabilization and increase the servicelifetime of the prosthesis. Fibroin solutions and suspensions can alsobe used to fill tissue defects such as those caused by bone tumorsurgery and reconstructive surgery, and to enhance bone and tissuerepair, for example to induce calcium precipitation, or osteoblastproliferation and activity, with the formation of newly formed bonetissue.

The fibroin solutions and suspensions comprising core/shell particlesare especially useful for bone repair. Without being held to theory, itis believed, in this case, that the bone repair process may occur asfollows. The first components to degrade may be the calcium ion-dopedshells of the core/shell particles, enabling body fluids to diffuse intothe calcium ion-rich water layers that serve as the interfacial zonebetween the biodegradable shells and the encapsulating fibroin web. Theporosity of the fibroin matrix and the additional porosity created bythe shell degradation enable the infiltration of osteoblast cells in theporous scaffold, which interact with the bioactive web of the scaffoldand initiate new tissue and bone growth. The next stage is presumablybiodegradation of the reactive layer, if present, for example byhydration of —SiO— bonds that bind the biodegradable shell to themineral core, thus introducing the resorbable mineral core into thefluid of the remaining hydrogel. The repair process is accompanied bybiodegradation of the fibroin as new bone is generated. The compositescaffold can maintain the mechanical stability of the three-dimensional,mechanically stable, high surface area web, at least during the firststages of bone regeneration.

A particularly advantageous feature of the fibroin solutions andsuspensions is that the material morphology and mechanical propertiesare readily varied by modification of the bioactive matrix and thecore/shell biodegradable particles to meet the requirements of specificapplications. It is contemplated that bioactive, biodegradable gels andpolymers other than fibroin can be used in combination with thecore/shell particles described herein to form scaffolds.

Fibroin solutions, suspensions, and composites can induce proliferationin such cell types as osteoblasts, keratinocytes, fibroblasts,pericytes, endothelial cells, and the like. Because fibroin solutionsand suspensions can enhance the proliferation of many cell types, theycan also be used in applications involving tissues other than bonetissue, for example, for soft tissue reconstruction such as afterplastic surgery, artificial skin applications, cartilage repair, and thelike.

The invention is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Purification of Silk Fibroin

Using techniques known in the art, cocoons of the Bombyx-mori silk wormwere first degummed. The glue-like sericin proteins were extracted byrepeated washing in aqueous solutions of sodium carbonate (Na₂CO₃), asfollows. The cocoons were first washed in an aqueous solution of 1.1 g/l(grams per liter) Na₂CO₃ at 98° C. for one hour, and then rinsed in anaqueous solution containing 0.4 g/l Na₂CO₃ at 98° C. for one hour. Thecocoons were then washed repeatedly in distilled water at temperaturesdecreasing from 98° C. to ambient, leaving behind sericin-free fibroin.The amount of raw fibroin in the water was 10 g/l. The degummed fibroinwas then dissolved at 65° C. in a 9.3 molar aqueous solution of lithiumbromide (LiBr) for 3 hours at an initial composition of 100 g fibroin/lsolution. When dissolution was complete, the aqueous solution wasdiluted to 5% weight/volume fibroin by the addition of distilled water.The remaining impurities and undissolved fiber were removed byfiltration, using a filter with pore size n.1. The salt was then removedfrom the fibroin mixture by 72 hours of dialysis against distilledwater, using a regenerated cellulose membrane with a molecular weightcut-off of 3,500. The final concentration of dialyzed fibroin was 1-2%pure silk fibroin.

Example 2 Formation of a Suspension by Thermal Treatment of an AqueousSolution of Fibroin

An aqueous fibroin solution (20 ml (milliliters)) was poured into apolystyrene capsule incubated at temperatures of −20° C. to 50° C. forperiods of 2 to 24 hours. The solutions incubated at −20° C. were heatedto and maintained at room temperature until gelation occurred. Otherfibroin solutions kept at constant temperatures from 4° C. to 50° C.,required incubation periods of 2 to 24 hours for gelation to occur. Thewater content of the gels, as determined by thermogravimetric analysis,varied from 95 to 98 wt %. The consistency of the suspensions wasdependent upon the time and temperature at which they were incubated.Thus, it was demonstrated that the Theological properties of the fibroincould be varied by thermal treatment.

Example 3 Enzymatic Treatment of the Silk Fibroin

The aqueous fibroin mixture was treated with various proteolytic enzymesthat are specific for reaction with bond sites of particular aminoacids. The goal was to reduce the average molecular weight, to increasethe biodegradation rate, and to expose specific active sites foreventual interaction with peptides specific for mediation with cellularadhesion factors, growth factors and pharmacological molecules. Thefollowing enzymes were studied:

Protease (from Streptomyces griseus): This enzyme hydrolyzes the peptidebonds at carboxylic sites of glutamic acid. It has been demonstratedthat peptide chains of fibroin having terminal end groups of glutamicacid impede the formation of the ordered beta structure of the silkfibroin. The enzymatic reactions were performed at enzyme-substrateconcentrations of 50 μl/ml (microliters/milliliter) and after one hourat 37° C. the solution becomes white in color and more viscous. The solthat is formed is an injectable material capable of forming a gel.Heating to 50° C. for 15 minutes deactivated the enzyme. The glutamicamino acid content within the fibroin protein chain was found to beapproximately 1%, as determined by electrophoretic analysis. There was anotable presence of peptides with lower molecular weight than waspresent in the fibroin mixture prior to treatment.

Papain: This enzyme is specific for reaction with chemical bonds ofleucine (0.5% of the fibroin) and glycine (44% of the fibroin). Theconditions of treatment and the enzyme-substrate concentration ratio canbe varied to produce different levels of viscosity of the resultingfibroin sol or solution. In this case, the product contains peptideswith molecular weights in the range of 20 Kd to 2 Kd, as determined byelectrophoretic analysis.

Chymotripsin: Treatment with this enzyme causes hydrolysis of thedisulfide bond that connects the Cp (heavy) fraction (composedprincipally of Glycine-Alanine-Serine) and the Cs fraction (light; amixture of short peptide chains) comprising the other amino acidspresent in the pure fibroin material. A buffered enzyme solution wasadded to the fibroin-water solution at 40° C. The degree of hydrolysiswas controlled by the time of reaction. Different treatment times couldtrigger different amounts of hydrolysis. At completion of the reaction,the resulting aqueous medium contained the Cs or light fraction insolution and a gelatinous precipitate of the Cp or heavy fraction.

Example 4 Formation of a Suspension by the Addition of Acids or Alcoholsto the Fibroin Solution

Gelation was induced by adding citric acid or glycerol to the fibroinsolution.

Citric acid was added drop by drop to 10 ml of fibroin solutions, untilthe pH reached 3.7 (the isoelectric point equals 3.8). Gelation occurredafter about 6 hours, producing a white, opaque porous gel that containedabout 95 wt % of water. FIG. 2 shows an environmental scanning electronmicrograph of the material formed.

When 3 ml of glycerol were added to 7 ml of fibroin solution, gelationoccurred in about 20 hours, producing a white translucent gel containingabout 90 wt % of water. FIG. 3 shows an environmental scanning electronmicrograph of the material formed.

Example 5 In Vitro Preparation of an Injectable FibroinGel/Hydroxyapatite/Poly(lactide) Composite Scaffold

A powder of core/shell particles of polymer-coated hydroxyapatiteparticles was made. A (poly)lactide (PLA) with a molecular weight ofapproximately 6,000 was used as the shell of biodegradable polymer. Twograms of silane-treated hydroxyapatite was added to 6 grams of apolylactide in 50 ml of butanol. The solution was heated to 70° C. for 2hours, cooled to room temperature and mixed with 50 ml of a 2%weight/volume aqueous solution of polyethylene glycol. The settling rateof the coated particles was slow enough to maintain relative uniformityof the coating process. The core/shell mixture was stirred, poured intoa glass Petrie dish, dried at 50° C. and lightly milled to a powder. Theresulting material was a rather broad size distribution of spherical,polymer-coated aggregates.

Two grams of the core/shell powder were mixed with 10 ml of fibroin gel.The powder dispersed readily in the gel to form paste. The pastecontaining core/shell particles was dried in two ways, namelyfreeze-dried and also slowly dried at room temperature. FIGS. 4 to 7 areoptical micrographs of the resulting scaffolds. Upon slow drying at roomtemperature, a particle-filled fibroin material with apparent structuralintegrity is formed. The freeze-dried sample exhibits the morphologicalstructure that one might expect, namely a broad distribution offibroin-coated.

Example 6 In Vitro Tests of Fibroin Suspensions

Human osteoblast-like cells MG63) were cultivated for 72 hours onfibroin gels made by adding glycerol to an aqueous fibroin solution andby treating the aqueous fibroin solution at 4° C. Biochemical andimmunoenzymatic parameters of MG63, grown on the tested materials and onan empty polystyrene well used as a control, were evaluated to determinecell proliferation and activity.

Cell proliferation on the pure fibroin gel prepared by thermal treatmentwas significantly lower when compared to the control, but, at the sametime, the gel favored osteoblast activity and differentiation, asdemonstrated by the enhanced ALP (alkaline phosphatase) activity(16.71+/−1.80 vs. control 10.10+/−1.61) and TGFβ1 (transforming growthfactor β1) levels, (487+/−29 vs. control 432+/−42), respectively. Cellproliferation on the gel prepared by adding glycerol showed littledifference when compared to the control.

Neither gel induced cytotoxicity, as revealed by the LDH (lactatedehydrogenase) level (pure fibroin gel, 14.67+/−2.04; gel with glycerol,18.03+/−0.23; control, 14.63+/−2.19).

Example 7 In Vivo Tests of Fibroin Suspensions

Citric acid derived fibroin gels were implanted in cavities (6 mmdiameter, 10 mm depth) drilled in the femoral condyle of rabbits.Implant-free cavities were used as a control. Rabbits were sacrificedafter a month from implantation, and histological results of the implantsites were compared to those of the implant-free sites.

After one month of implantation, the hole in one cavity filled by thefibroin gel was completely filled by newly formed bone (FIG. 8A). Nearlycomplete bone healing with some residual fibroin 4 was observed. Thecavity with no fibroin added displayed no bone regeneration (see FIG.8B). The original bone 5 and the empty defect 6 were observed. In fourof the cavities filled with the fibroin gel, the cavities were nearlyfilled with newly formed bone 7, with small residual cavities 8 beingstill present (FIG. 9A). In FIG. 9A, the cavity has a width of 1.08millimeters and a length of 1.7 millimeters. FIG. 10 shows the interfacebetween the original bone 5 and the newly grown bone 7. The new boneformed well-organized trabeculae that propagated from the original bonetrabeculae without creating any gap between the newly-formed and theoriginal bone. A residual cavity 8 was observed as was connective tissueundergoing mineralization 9. A mild inflammation was observed, with theabsence of any granuloma and apparent adverse immunological response.Only fragments of the original fibroin gel were still visible,confirming the ability of the gel to degrade at a rate that iscompatible with the rate of tissue regeneration, thus providing evidencethat the fibroin suspensions are useful for the proposed applications.

Novel fibroin suspensions and composites, have been disclosed. Suchfibroin suspensions can be made from a solution of purified fibroinusing heat, proteolytic enzymes, acids, alcohols, or a biocompatiblepolymer. The fibroin suspensions can be in the form of occlusions, gels,creams or pastes. The fibroin suspensions and composites as well assolutions have particular utility as for use in bone and tissueconstruction, repair and regeneration. Because the fibroin solutions,suspensions, and composites can be injected, the need for many invasivesurgical procedures, particularly orthopedic procedures can be reduced,thus minimizing the potential for infection decreasing the requiredrecovery periods and lowering the overall cost of the medicalprocedures.

While the invention has been described with reference to a preferredembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing fromessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

1. A method to induce cell proliferation, comprising contacting a cellwith a fibroin solution or suspension in an amount effective to inducecell proliferation, wherein the fibroin solution or suspension isinjectable and bioactive.
 2. The method of claim 1, wherein the fibroinsuspension is in the form of an occlusion, a gel, a cream, or a paste.3. The method of claim 1, wherein the fibroin solution or suspension isinjectable through a syringe needle having a diameter of 584micrometers, 838 micrometers, or 2 millimeters.
 4. The method of claim1, wherein the fibroin solution or suspension forms a matrix or scaffoldat the site for cell proliferation, wherein the matrix or scaffoldpermits infiltration and growth of a cell to be proliferated.
 5. Themethod of claim 1, wherein the fibroin is in the form of a suspensionwith at least one phase comprising a solvent and at least one phasecomprising the fibroin.
 6. The method of claim 1, wherein the fibroinsuspension further comprises at least one physiologically active agent.7. The method of claim 1, wherein the cell comprises an osteoblast, akeratinocyte, a fibroblast, a pericyte, a chondrocyte, or an endothelialcell.
 8. The method of claim 1, wherein the cell proliferation is usedfor soft tissue reconstruction.